The goal of this project is to understand in mechanistic terms how proteins are transported across membranes. Both secretory and membrane proteins are translocated from the cytosol across the membrane through a channel that is formed from a heterotrimeric membrane protein complex, the Sec61p complex in eukaryotes and the SecY complex in bacteria and archae. We have determined X-ray structures of the SecY complex alone and when associated with the ATPase SecA, which have led to new insights and provide the basis for part of the present proposal. In eukaryotes, there is a translocation pathway in the reverse direction, called ERAD (for ER associated degradation), which is used to degrade misfolded ER proteins. We have identified most, if not all, components involved in ERAD, paving the way for mechanistic studies. Here, we will address key aspects of translocation with specific emphasis on the following questions: 1. How are proteins cotranslationally translocated and how is the membrane barrier for small molecules maintained during the process? Based on a new method to generate cotranslational translocation intermediates in intact E. coli cells and the ability to purify ribosome/nascent chain/channel complexes, we will determine how many copies of SecY are required for translocation and will use electron microscopy to elucidate how the active channel binds to the ribosome. We will investigate how the channel maintains the membrane barrier for small molecules during translocation. 2. What is the mechanism of posttranslational translocation in bacteria? We will clarify the mechanism by which SecA moves polypeptides through the channel. We will address the unexplored role of the SecDFYajC complex and test its involvement in mediating the effect of a membrane potential on translocation. 3. What is the molecular mechanism of ERAD? We will probe the path of a luminal ERAD (ERAD-L) substrate and determine how it is recognized. Based on preliminary results that indicate a crucial role for the ubiquitin ligase Hrd1p, we will purify the protein, and reconstitute it together with its partner proteins. We will develop a purified component system that recapitulates subreactions or even the entire ERAD-L process. PUBLIC HEALTH RELEVANCE: The mechanism of protein translocation is of great medical importance. Drugs that inhibit signal sequence binding can be used for therapeutic intervention in chronic inflammatory diseases. A large number of diseases, including cystic fibrosis and a1-antitrypsin deficiency, are caused by mutations that result in the misfolding of ER proteins and their subsequent degradation in the cytosol. The pathway is also hijacked by certain viruses and toxins, and a better understanding may lead to new drugs allowing interference.